There are hundreds of pathological mutants in mice and rats, and many of these are of interest to biomedical research. Mutant mice are typically used for specialized research and development projects. Mutant mice can be maintained with either outbred or inbred genetic backgrounds, the properties of which confer genetic advantages and disadvantages. For example, the athymic nude gene on the outbred HsdCpb:NMRI background produces a relatively robust animal, but the same mutation in mice having a BALB/cOlaHsd background produces a less robust animal.
Prkdcscid is a mutation of the gene encoding the catalytic subunit of DNA activated protein kinase (Prkdc) that results in a severe combined immunodeficiency (scid) (for review, see Bosma and Carroll, 1991). It is an autosomal recessive mutation that disrupts the differentiation of B-cell and T-cell progenitor cells due to a defective variable, diversity and joining (VDJ) recombinase system necessary for generating B- and T-cell receptors. Mice with a Prkdc mutation lack Thy-1-positive dendritic epidermal cells. Unlike nude mice, Prkdcscid mutant mice posses a thymus and lymph nodes.
The non-obese diabetic (NOD) mouse is an animal model of Type 1 diabetes, exhibiting a susceptibility to spontaneous development of automimmune, T cell-mediated insulin-dependent diabetes mellitus (IDDM). The NOD strain and related strains were developed at Shionogi Research Laboratories in Aburahi, Japan, by Makino and colleagues and first reported in 1980 (Makino et al., 1980). Additionally, NOD strains are typically characterized by a functional deficit in natural killer (NK) cells, defects in myeloid development and function as well as in the differentiation and function of antigen presenting cells (APCs), and a C5 deficiency that inhibits activation of both classical and alternative pathways of hemolytic complement (Greiner et al., 1998).
Mouse strains carrying a Prkdcscid mutation, while displaying a lack of B- and T-cell function, express normal levels of NK cells, hemolytic complement and myeloid function. In addition, Prkdcscid mutants on various background strains produce immunoglobulin and functional T-cells at low levels as the mice age, a phenomenon referred to as “leakiness.” These innate immune properties are disadvantageous for certain research applications, especially engraftment of human tumor cell lines.
The Prkdcscid mutation has been transferred from the C.B-17 background onto the diabetes susceptible nonobese diabetic (NOD) background and is referred to as the NOD scid. Unlike the typical NOD mouse, development of autoimmune diabetes does not occur due to lack of T-cell function. NOD scid mice are further characterized by a NK cell functional deficit, macrophage deficiency, and a lack of detectable hemolytic complement (Greiner et al., 1998).
In addition, the “leaky” phenotype of homozygous scid mice, resulting from the incomplete blocking of VDJ recombinase activity that leads to residual B- and T-cell activity, is suppressed in this model compared to other scid mice on various genetic backgrounds. These characteristics are advantageous for studies involving long-term growth of transplanted cells. For example, human cell lines such as PC-3 and DU145, and CD-34 positive stem cells may effectively be grown long-term in NOD scid mice, but not in other scid mice that exhibit normal NK cell function, macrophages, and circulating complement that may influence tumor growth or engraftment of human stem cells. The immunologic profile of the NOD scid mouse renders it useful for biomedical research in oncology, immunology, hematology, HIV pathology, and other fields.
Despite their suitability for numerous applications, NOD scid mice exhibit some characteristics that can be disadvantageous for certain research purposes. For example, NOD scid mice have a full coat of albino hair, so their hair must be shaved prior to inoculation of cells, and subsequent visualization and imaging of tumors is more difficult than in a hairless model. Further, even after shaving a haired mouse, hair follicles remain which may autofluoresce and interfere with imaging studies employing bioluminescent and fluorescent reporters. The hairless homozygous mutant, unlike the nude mouse, is devoid of hair follicles (Lyon et al., 1996) thereby eliminating the autofluorescence from follicles that occurs in other xenograft models.
Described herein are improvements to the NOD scid mouse made by crossing NOD scid mice with a mouse carrying a mutation in the hairless gene (Hr) and backcrossing onto the NOD scid genetic background to produce a hairless NOD scid mouse strain. The hairless NOD scid mice disclosed herein are particularly advantageous for the studies of xenograft transplantation, spontaneous tumors, cancer cell tumorigenesis, tumor angiogenesis, tumor metastatic potential, tumor suppression therapy, carcinogenesis regulation, tumor imaging, and human stem cell engraftment.
The following various embodiments are contemplated:
1) A hairless, immunodeficient mouse on a non-obese diabetic (NOD) background having B-cell, T-cell, NK-cell, macrophage and complement deficiency.
2) The mouse of clause 1, wherein the mouse is homozygous for a Prkdc allele.
3) The mouse of clause 2, wherein the Prkdc allele is scid (Prkdcscid).
4) The mouse of any of clauses 1 to 3, wherein the mouse is homozygous for an Hr allele.
5) The mouse of clause 4, wherein the Hr allele is hr (Hrhr).
6) The mouse of any of clauses 1 to 5, wherein the non-obese diabetic background is NOD.CB17-Prkdcscid.
7) The mouse of any of clauses 1 to 5, wherein the non-obese diabetic background is NOD.CB17-Prkdcscid/NCrHsd.
8) The hairless mouse of any of clauses 1 to 7 further having a dendritic cell deficiency, wherein the dendritic cell deficiency is a reduction in the number of dendritic cells compared to an immunodeficient mouse of the same background that is not hairless.
9) A method of producing a hairless, immunodeficient mouse on a non-obese diabetic (NOD) background, the method comprising:
(a) crossing a NOD scid mouse strain homozygous for a Prkdc allele with a second mouse strain homozygous for an Hr allele to produce progeny heterozygous for both the Hr allele and the Prkdc allele;
(b) intercrossing the heterozygous progeny produced from step (a) with NOD scid mice;
(c) selecting offspring of step (b) that are homozygous for the Prkdc allele and have a genotype that is more similar to NOD scid;
(d) crossing the offspring selected in step (c) with mice homozygous or heterozygous for the Hr allele.
10) The method of clause 9 wherein the Prkdc allele is scid (Prkdcscid).
11) The method of any of clauses 9 to 10 wherein the Hr allele is hr (Hrhr).
12) The method of any of clauses 9 to 11, wherein the progeny produced from step (a) are genotyped using a process selected from the group consisting of:
single nucleotide polymorphism allelic discrimination, polymerase chain reaction, and single nucleotide polymorphism profiling.
13) The method of any of clauses 9 to 12, wherein the NOD scid mouse of steps (a) and (b) is a NOD.CB17-Prkdcscid mouse.
14) The method of any of clauses 9 to 12, wherein the NOD scid mouse of steps (a) and (b) is a NOD.CB17-Prkdcscid/NCrHsd-Prkdcscid mouse.
15) The method of any of clauses 9 to 14 wherein the second mouse strain of step (a) is an MF-1-hr mouse.